Urokinase is an enzyme involved in the metastasis of tumor cells, neovascularization, and other activities. One purpose of the present invention is to provide novel compounds which are active as inhibitors of urokinase that can be used to inhibit the activity of urokinase and thereby attenuate its deleterious effects. Another purpose of the present invention is to provide novel compounds which inhibit blood vessel formation, particularly blood vessel formation related to a pathologic condition.
Urinary-type plasminogen activator (uPA; urokinase) is a serine protease within the trypsin/chymotrypsin family. In its physiological state, uPA is found in three forms: single chain pro-uPA, two chain uPA, and low molecular weight uPA (lacks N-terminal domains). The zymogen, pro-uPA, is converted to u-PA by cleavage of the peptide bond at K158-I159. The resultant two chain uPA is linked by disulfide bridges, has an Mr of about 50 kD, and a C-terminal serine proteinase domain.
The activity of uPA is focused to cell surfaces upon binding to its receptor, uPAR. uPAR is a single-chain glycosyl phosphatidyl inositol (GPI)-anchored membrane receptor. The N-terminal 92 amino acids of uPAR play a dominant role in binding to uPA and pro-uPA. Receptor for uPA has been located on T-cells, NK cells, monocytes, and neutrophils, as well as vascular endothelial cells, fibroblasts, smooth muscle cells, keratinocytes, placental trophoblasts, hepatocytes, and a wide variety of tumor cells.
After conversion of pro-uPA to uPA, which occurs primarily at the uPAR on the cell surface, uPA activates plasminogen to plasmin. Activation occurs upon cleavage at residues PGR-VV for human plasminogen, or at residues SGR-IV for bovine plasminogen. Because plasminogen also is present on the cell surface, this activation cascade focuses the activity of u-PA and plasmin on the plasma membrane. Plasmin has many roles, including activation of additional uPA and other enzymes, digestion of fibrin, and digestion of components of the extracellular matrix (ECM). Digestion of the ECM surrounding a tumor removes the ECM as a physical barrier to metastasizing cells, which are then free to leave primary tumors and invade secondary sites. A review of the role of the uPA/uPAR system in cancer metastasis is provided in xe2x80x9cThe Urokinase-type Plasminogen Activator System in Cancer Metastasis: A Reviewxe2x80x9d, Andreasen et al., Int. J. Canc. 72:1-22 (1997).
A correlation between a high level of uPA and a high rate of metastasis, and poor prognosis, has been noted in certain tumors, especially breast cancer [Quax et al., J. Cell Biol. 115:191-199 (1991); Duffy et al., Cancer Res. 50:6827-6829 (1990)]. For instance, tumors of the lung [Oka et al., Cancer Res. 51:3522-3525 (1991)], bladder [Hasui et al., Int. J. Cancer 50:871-873 (1992)], stomach [Nekarda et al., Lancet 343:117 (1994)], cervical cancer [Kobayashi et al., Cancer Res. 54:6539-6548 (1994)], ovary [Kuhn et al., Gynecol. Oncol. 55:401-409 (1994)], kidney [Hofmann et al., Cancer 78:487-492 (1996)], brain [Bindahl et al., J. Neuro-Oncol. 22:101-110 (1994)], and soft tissue sarcoma [Choong et al., Int. J. Cancer (Pred. Oncol.) 69:268-272 (1996)] have exhibited a high level of uPA and/or uPA activity and a high rate of metastases. Overproduction of uPA has been reported to result in increased skeletal metastasis by prostate cancer cells in vivo [Achbarou et al., Cancer Res. 54:2372-2377 (1994)].
Inhibition or lowering of uPA activity, or disruption/inhibition of the interaction between uPA and its receptor (uPAR) has been shown to have a positive effect on maintenance of the extracellular matrix and an inhibitory effect on metastasis [Ossowski and Reich, Cell 35:611-619 (1983); Ossowski, Cell 52:321-328 (1988); Ossowski, J. Cell Biol. 107:2437-2445 (1988); Wilhelm et al., Clin. Exp. Metastasis 13:296-302 (1995); Achbarou et al., Cancer Res. 54:2372-2377 (1994); Crowley et al., Proc. Natl. Acad. Sci. USA 90:5021-5025 (1993); Kook et al., EMBO J. 13:3983-3991 (1994)]. The results of such experimental studies suggest that uPA-catalyzed plasminogen activation is rate-limiting for tumor progression, local tumor invasion and/or formation of distant metastasis. [Andreasen et al., Int. J. Canc. 72:1-22 (1997)].
The effects of the uPA system on cell migration and invasion are thought to be due to both a proteolytic effect of plasmin-mediated degradation of the extracellular matrix, as well as more a direct interaction of the uPA receptor with components of the extracellular matrix. Degradation of the extracellular matrix permits a metastasizing cell to invade the matrix, whereas interaction between uPA receptor and the matrix itself assists a cell in its migration. Localization of the uPA/plasmin system on the cell surface, or the leading edge of metastasizing cells, is consistent with postulated role of uPA in metastasis [Plesner et al., Stem Cells 15:398-408 (1997)].
Interaction of uPAR with vitronectin, a component of the extracellular matrix, mediates cell adhesion and can be enhanced when uPAR is bound by uPA. Cell surface adhesion molecules, integrins, also appear to be involved in this adhesion function, particularly beta-1 and beta-2 integrins [Paysant et al., Br. J. Haematol. 100:45-51 (1998); Simon et al., Blood 88:3185-3194 (1996)]. The CD11b/CD18 integrin can associate with the uPA-uPAR complex and promote adhesion of cells bearing these receptors, e.g., neutrophils, leukocytes.
The uPA/uPAR system also is involved in the establishment of new vasculature, or neovascularization.
Establishment of new vasculature is required for sustaining primary and metastatic tumor growth. Pathological neovascularization also is a characteristic of retinal disease, rubeosis iritis, proliferative vitreo retinopathy inflammatory disease, diabetic retinopathy, chronic uveitis, Fuch""s heterochromic iridocyclitis, neovascular glaucoma, corneal or optic nerve neovascularization, vascular disease, pterygium, glaucoma surgery bleb failure, hyperkeratosis, cheloid and polyp formation (see EP 451,130). Undesired angiogenesis also can occur in the following conditions or can be a result of the following activities: macular degeneration, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sogrens disease, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections other than leprosy, lipid degeneration, chemical burns, bacterial or fungal ulcers, Herpes simplex or zoster infections, protozoan infections, Kaposi""s sarcoma, Mooren ulcer, Terrien""s marginal degeneration, marginal keratolysis, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegeners sarcoidosis, sleritis, Steven""s Johnson disease, radial keratotomy, sickle cell anemia, sarcoid, pseudoxanthoma elasticum, Pagets disease, vein or artery occlusion, carotid obstructive disease, chronic uveitis, chronic vitritis, Lyme""s disease, Eales disease, Bechets disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, post-laser complications, abnormal proliferation of fibrovascular tissue, hemangiomas, Osler-Wever-Rendu, solid tumors, blood borne tumors, AIDS, ocular neovascular disease, osteoarthritis, chronic inflammation, Crohn""s disease, ulcerative colitis, tumors of rhabdomyosarcoma, tumors of retinoblastoma, tumors of Ewing sarcoma, tumors of neuroblastoma, tumors of osteosarcoma, leukemia, psoriasis, atherosclerosis, pemphigoid, as recited in U.S. Pat. No. 5,712,291.
An antagonist of uPA/uPAR binding (EGF-like domain of uPA fused to Fc of IgG) was said to inhibit neovascularization and growth of the murine B16 melanoma. [Min et al., Cancer Res. 56:2428-2433 (1996)]. Consistent with this finding is the correlation noted between microvessel density, vascular invasion and uPA levels in breast carcinomas [Hildenbrand et al., Brit. J. Cancer 72:818-823 (1995)]. The known uPA inhibitor amiloride also was said to inhibit a variety of neovascularization pathologies [Glaser et al., EP 451,130; Avery et al., Arch. Ophthalmol. 108:1474-1476 (1990)].
There are two primary physiological inhibitors of uPA, PAI-1 and PAI-2, which are members of the serpin family of proteinase inhibitors. The binding of serpins to their cognate proteases involves a large number of interactions between amino acids of each protein, including those in the serpin reactive loop (Ser-Ala-Arg-Met-Ala (SEQ. ID. NO. 1) for PAI-1, Thr-Gly-Arg-Thr-Gly (SEQ. ID. NO. 2) for PAI-2). Introduction of exogenous PAI-2 into experimental animals was reported to inhibit the rate of lung metastasis [Evans and Lin, Amer. Surg. 61:692-697 (1995); Mueller et al., Proc. Natl. Acad. Sci. USA 92:205-209 (1995)]. The ability of PAI-1 to inhibit metastasis has not yet been consistently shown. The gene for PAI-1, and means for its recombinant expression, are disclosed in Loskutoff et al., U.S. Pat. No. 4,952,512. Recombinant and native human PAI-2 is disclosed in Stephens et al., U.S. Pat. No. 5,422,090.
The most widely studied uPA inhibitors may be within the 4-substituted benzo[b]thiophene-2-carboxamidine class of inhibitors, of which B428 (4-iodo-benzo[b]thiophene-2-carboxamidine) and B623 are members [Towle et al., Cancer Res. 53:2553-2559 (1993); Bridges et al., Bioorg. Med. Chem. 1:403-410 (1993); Bridges et al., U.S. Pat. No. 5,340,833]. Infusion of B428 in experimental rats inoculated with tumor cells was said to inhibit uPAR gene expression, decrease the primary tumor volume and decrease metastases [Xing et al., Cancer Res. 57:3585-3593 (1997)]. Daily intraperitoneal treatment of mice bearing tumors with B428 or B623 was said to block metastasis to muscle and fat, but did not inhibit tumor-induced angiogenesis or reduce the rate of spontaneous lung metastasis. In fact, B623 enhanced the formation of lung metastasis (Alonso et al., Breast Cancer Res. Treat. 40:209-223 (1996)]. Infusion of B428 in a syngeneic model of rat prostate cancer also lead to a decrease in primary tumor volume and tumor weight, and a decrease in metastasis [Rabbani et al., Int. J. Cancer 63:840-845 (1995)].
Other known inhibitors of uPA include p-aminobenzamidine, which is a competitive inhibitor of uPA, and amiloride. Both compounds have been shown to reduce tumor size in experimental animals [Jankan et al., Cancer Res. 57:559-563 (1997); Billstrom et al., Int. J. Cancer 61:542-547 (1995)]. Recently, epigallo-cathecin-3 gallate (EGCG), a polyphenol found in green tea, was reported to bind uPA and inhibit its activity [Jankun et al., Nature 387:561 (1997)]. Those researchers concluded EGCG is a weaker inhibitor of uPA than amiloride, but suggested EGCG can be consumed in much higher doses than amiloride without toxic effect. A competitive inhibitor of uPA, xcex1-N-benzylsulfonyl-p-aminophenylalanine, is disclosed by Pye et al. in U.S. Pat. No. 4,165,258.
Other approaches at inhibiting the uPA/uPAR system include development of a bifunctional hybrid molecule consisting of the uPAR-binding domain of uPA and PAI-2, which is said to inhibit uPA and bind uPAR in vitro [Ballance et al., Eur. J. Biochem. 207:177-183 (1992)]. Antagonists of uPAR also have been studied [Doyle and Rosenberg, U.S. Pat. No. 5,656,726; Min et al., Cancer Res. 56:2428-2433 (1996)], as have antisense oligonucleotides complementary to uPA [Wilhelm et al., Clin. Exp. Metast. 13:296-302 (1995); Iversen and Scholar, U.S. Pat. No. 5,552,390]. Antibodies directed against uPAR, and said to inhibit the binding of uPA to uPAR, are disclosed by Dano et al. in U.S. Pat. No. 5,519,120. Small molecules said to inhibit urokinase, along with a variety of other serine proteases, include those disclosed by Abe et al. in U.S. Pat. No. 5,508,385 and U.S. Pat. No. 5,153,176, and by Takano et al. in J. Pharmacol. Exp. Therapeut. 271:1027-1033 (1994).
Compounds have been developed to directly inhibit the binding of u-PA to uPAR (Crowley et al., Proc. Natl. Acad. Sci. USA 90:5021-5025 (1993); Goodson et al., Proc. Natl. Acad. Sci. USA 91:7129-7133 (1994); Kobayashi et al., Brit. J. Cancer 67:537-544 (1993), and Int. J. Cancer 57:727-73f3 (1994), and J. Biol. Chem. 270:8361-8366 (1995); Lu et al., FEBS Lett. 356:56-59 (1994) and FEBS Lett. 380:21-24 (1996)]. Additionally, pro-hepatocyte growth factor (HGF), a cell migration stimulating protein, is a substrate of uPA [Naldinie et al., EMBO J. 11:4825-4833 (1992)]. Direct cleavage of a 66 kDa extracellular matrix protein and fibronectin by uPA also has been reported, which suggests a more direct role for uPA in facilitating cell migration [Quigley et al., Proc. Natl. Acad. Sci. 84:2776-2780 (1987)]. Thus, inhibition of uPA may affect these activities, as well.
The present invention is directed to novel pepitidic non-covalent urokinase inhibitors. The compounds have an arginine mimic at P1. These compounds have activity as potent inhibitors of urokinase and thereby are useful in decreasing its deleterious effects. Compounds of the present invention are active in inhibiting blood vessel formation, particularly that related to a pathologic process.
Thus in one aspect, the present invention is directed to compounds of the formula (I): 
wherein:
(a) X is selected from the group consisting of xe2x80x94S(O)2xe2x80x94, xe2x80x94N(Rxe2x80x2)xe2x80x94S(O)2xe2x80x94, xe2x80x94(Cxe2x95x90O)xe2x80x94, xe2x80x94OC(xe2x95x90O)xe2x80x94, xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94P(O)(Rxe2x80x2)xe2x80x94, and a direct link, wherein Rxe2x80x2 is independently hydrogen, alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms or aralkyl of about 7 to about 16 carbon atoms, with the proviso that when X is xe2x80x94P(O)(Rxe2x80x2)xe2x80x94, then Rxe2x80x2 is not hydrogen;
(b) R1 is selected from the group consisting of:
(1) alkyl of 1 to about 12 carbon atoms which is optionally substituted with Y1 and/or Y2,
(2) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of about 3 to about 8 carbon atoms which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(3) cycloalkyl of 3 to about 15 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(4) heterocycloalkyl of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S(O)i, wherein i is 0, 1 or 2, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(5) heterocyclo of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S(O)i, wherein i is 0, 1, or 2, including, 
xe2x80x83wherein 
xe2x80x83is a 5 to 7 member heterocycle having 3 to 6 ring carbon atoms, where V is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)xe2x80x94, xe2x80x94S(O)2xe2x80x94 or xe2x80x94Sxe2x80x94, which is optionally mono-, di-, or tri-substituted on the ring carbons with Y1, Y2, and/or Y3,
(6) alkenyl of 2 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 3 to about 8 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(7) aryl of about 6 to about 14 carbon atoms which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(8) heteroaryl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(9) aralkyl of about 7 to about 15 carbon atoms which is optionally substituted on the alkyl chain with hydroxy or halogen and which is optionally mono-, di-, or tri-substituted on the aryl ring with Y1, Y2, and/or Y3,
(10) heteroaralkyl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, which is optionally substituted on the alkyl chain with hydroxy or halogen and which is optionally mono-, di- or tri-substituted on the ring with Y1, Y2, and/or Y3,
(11) aralkenyl of about 8 to about 16 carbon atoms which is optionally mono-, di-, or tri-substituted on the aryl ring with Y1, Y2, and/or Y3,
(12) heteroaralkenyl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di- or tri-substituted on the ring carbons with Y1, Y2, and/or Y3, 
(17) fused carbocyclic alkyl of about 9 to about 15 carbon atoms;
(18) difluoromethyl or perfluoroalkyl of 1 to about 12 carbon atoms,
(19) perfluoroaryl of about 6 to about 14 carbon atoms,
(20) perfluoroaralkyl of about 7 to about 15 carbon atoms, and
(21) hydrogen when X is a direct link; wherein each Y1, Y2, and Y3 is independently selected and is
(i) selected from the group consisting of halogen, cyano, nitro, tetrazolyl, guanidino, amidino, methylguanidino, xe2x80x94CF3, xe2x80x94CF2CF3, xe2x80x94CH(CF3)2, xe2x80x94C(OH) (CF3)2, xe2x80x94OCF3, xe2x80x94OCF2H, xe2x80x94OCF2CF3, xe2x80x94OC(O)NH2, xe2x80x94OC(O)NHZ1, xe2x80x94OC(O)NZ1Z2, xe2x80x94NHC(O)Z1, xe2x80x94NHC(O)NH2, xe2x80x94NHC(O)NZ1, xe2x80x94HC(O)NZ1Z2, xe2x80x94C(O)OH, xe2x80x94C(O)OZ1, xe2x80x94C(O)NH2, xe2x80x94C(O)NHZ1, xe2x80x94C(O)NZ1Z2, xe2x80x94P(O)3H2, xe2x80x94P(O)3(Z1)2, xe2x80x94S(O)3H, xe2x80x94S(O)mZ1, xe2x80x94Z1, xe2x80x94OZ1, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NHZ1, xe2x80x94NZ1Z2, xe2x80x94N-morpholino, and xe2x80x94S(O)m(CF2)qCF3, wherein m is 0, 1 or 2, q is an integer from 0 to 5, and Z1 and Z2 are independently selected from the group consisting of alkyl of 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 ring atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms, or
(ii) Y1 and Y2 are selected together to be xe2x80x94O[C(Z3)(Z4)]rOxe2x80x94 or xe2x80x94O[C(Z3)(Z4)]r+1xe2x80x94, wherein r is an integer from 1 to 4 and Z3 and Z4 are independently selected from the group consisting of hydrogen, alkyl of 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 ring atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms;
(c) R2 is selected from the group consisting of xe2x80x94CH3, xe2x80x94C2H5, xe2x80x94(CH2)2OH, xe2x80x94(CH2)2OA1, xe2x80x94CH(R5) OH, xe2x80x94CH (R5) OA1 and xe2x80x94CH2NHxe2x80x94Xxe2x80x2 xe2x80x94R6 wherein A1 is xe2x80x94C(xe2x95x90O)OR6, xe2x80x94C(xe2x95x90O)R6 or xe2x80x94C(xe2x95x90O)NR5R6; Xxe2x80x2 is selected from the group consisting of xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94N(Rxe2x80x3)xe2x80x94, xe2x80x94(Cxe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94, xe2x80x94P(O) (Rxe2x80x3)xe2x80x94, and a direct link, wherein Rxe2x80x3 is hydrogen, alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms or aralkyl of about 7 to about 16 carbon atoms with the proviso that when Xxe2x80x2 is xe2x80x94P(O)(Rxe2x80x3)xe2x80x94, then Rxe2x80x3 is not hydrogen; R5 is selected from the group consisting of:
(1) alkyl of 1 to about 4 carbon atoms, optionally substituted with Y1 and/or Y2,
(2) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of 3 to about 6 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(3) cycloalkyl of 3 to about 6 carbon atoms, which is optionally mono-, di-, or trisubstituted on the ring with Y1, Y2, and/or Y3,
(4) heterocycloalkyl of 4 to about 6 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S(O)i, wherein i is 0, 1 or 2, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(5) heterocyclo of 4 to about 6 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S(O)i, wherein i is 0, 1, or 2, including 
xe2x80x83wherein 
xe2x80x83is a 5 to 6 member heterocycle having 3 to 5 ring carbon atoms, where V is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)xe2x80x94, xe2x80x94S(O)2xe2x80x94 or xe2x80x94Sxe2x80x94, which is optionally mono-, di-, or tri-substituted on the ring carbons with Y1, Y2, and/or Y3,
(6) alkenyl of 2 to about 6 carbon atoms which is optionally substituted with cycloalkyl of 3 to about 6 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(7) phenyl which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(8) heteroaryl of about 5 to about 6 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(9) alkyl of 1 to about 4 carbon atoms which is substituted with phenyl and which is optionally mono-, di-, or tri-substituted on the phenyl ring with Y1, Y2, and/or Y3,
(10) heteroaralkyl of about 5 to about 6 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally substituted on the alkyl chain with hydroxy or halogen and optionally mono-, di- or tri-substituted on the ring with Y1, Y2, and/or Y3,
(11) aralkenyl of about 8 to about 12 carbon atoms which is optionally mono-, di-, or tri-substituted on the aryl ring with Y1, Y2, and/or Y3,
(12) heteroaralkenyl of 5 to about 6 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di- or tri-substituted on the ring carbons with Y1, Y2, and/or Y3, and
(13) hydrogen; and R6 is selected from the group consisting of:
(1) alkyl of 1 to about 12 carbon atoms, optionally substituted with Y1 and/or Y2,
(2) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of 3 to about 8 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(3) cycloalkyl of 3 to about 15 carbon atoms, which is optionally mono-, di-, or trisubstituted on the ring with Y1, Y2, and/or Y3,
(4) heterocycloalkyl of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S(O)i, wherein i is 0, 1 or 2, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2, and/or Y3,
(5) heterocyclo of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S(O)i, wherein i is 0, 1, or 2, including 
xe2x80x83wherein 
xe2x80x83is a 5 to 7 member heterocycle having 3 to 6 ring carbon atoms, where V is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)xe2x80x94, xe2x80x94S(O)2xe2x80x94 or xe2x80x94Sxe2x80x94, which is optionally mono-, di-, or tri-substituted on the ring carbons with Y1, Y2, and/or Y3,
(6) aryl of about 6 to about 14 carbon atoms which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(7) heteroaryl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di- or tri-substituted with Y1, Y2, and/or Y3,
(8) aralkyl of about 7 to about 15 carbon atoms which is optionally mono-, di-, or tri-substituted on the aryl ring with Y1, Y2, and/or Y3,
(9) heteroaralkyl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally substituted on the alkyl chain with hydroxy or halogen and optionally mono-, di- or tri-substituted on the ring with Y1, Y2, and/or Y3, and
(10) hydrogen, with the proviso that R6 is not hydrogen when A1 is xe2x80x94C(xe2x95x90O)OR6;
(d) R3 is selected from H or methyl, or R3 and R4 are selected together as set forth in (f);
(e) R4 is in the S configuration and is selected from the group consisting of H, xe2x80x94CH2xe2x80x94Sxe2x80x94CH3, xe2x80x94CH2OH, xe2x80x94CH2CN, lower alkyl of 1 to about 3 carbon atoms, xe2x80x94CH2Cxe2x89xa1CH, xe2x80x94CH2CHxe2x95x90CH2 and xe2x80x94CHxe2x95x90CH2 or R3 and R4 are selected together as set forth in (f);
(f) alternatively, R3 and R4 are selected together to be in the S configuration to give a group at P2 selected from the group consisting of prolyl, pipecolyl, azetidine-2-carbonyl, 4-hydroxyprolyl, 3-hydroxyprolyl, 3,4-methanoprolyl, and 3,4-dehydroprolyl;
(g) R7 is hydrogen or alkyl of 1 to about 4 carbon atoms; and
(h) E is selected from 
xe2x80x83wherein R8 and R9 are independently selected from hydrogen, hydroxy, halogen, alkyl of 1 to about 4 carbon atoms, alkyl of 1 to about 4 carbon atoms substituted with alkoxy of 1 to about 4 carbon atoms, alkoxy of 1 to about 6 carbon atoms, and trifluoromethyl; R10 and R11 are independently hydrogen, hydroxy, alkoxy of 1 to about 3 carbon atoms, trihydrocarbylsilyl of 3 to about 16 carbon atoms, alkyl of 1 to about 3 carbon atoms or xe2x80x94C(xe2x95x90O)R12; R12 is hydrogen, alkyl of 1 to about 6 carbon atoms, alkoxy of 1 to about 6 carbon atoms or (CF2)jCF3 wherein j is 0, 1, 2 or 3 with the proviso that R10 and R11 are not both hydroxy or alkoxy; each of R13 and R14 is independently selected from hydrogen or lower alkyl of 1 to about 3 carbon atoms; and t is an integer from 0 to 6; and pharmaceutically acceptable salts thereof.
The compounds of the present invention can be divided into parts termed P1, P2, P3 and P4 as shown in the following formula Ia: 
wherein X, R1, R2, R3, R4, R7 and E are as defined in connection with formula (I). Thus, the portion of a compound of formula (I) referred to as P1 or P1 is the moiety 
The portion of a compound of formula (I) ref erred to as P2 or P2 is the moiety 
The portion of a compound of formula (I) referred to as P3 or P3 is the moiety 
Among other factors, the present invention is based on our finding that the novel compounds of our invention are active as inhibitors of urokinase. Compounds of the present invention exhibit activity in inhibiting angiogenesis.
In another aspect, the present invention is directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention and a pharmaceutically acceptable carrier.
In yet another aspect, the present invention is directed to methods of using the compounds and pharmaceutical compositions of the present invention for inhibition of urokinase.
Definitions
In accordance with the present invention and as used herein, the following terms are defined to have following meanings, unless explicitly stated otherwise:
The term xe2x80x9calkenylxe2x80x9d refers to unsaturated aliphatic groups having at least one double bond.
The term xe2x80x9calkylxe2x80x9d refers to saturated aliphatic groups including straight-chain, branched-chain and cyclic (including polycyclic) groups.
The terms xe2x80x9calkoxyxe2x80x9d and xe2x80x9calkoxylxe2x80x9d refer to a group having the formula, Rxe2x80x94Oxe2x80x94, wherein R is an alkyl group.
The term xe2x80x9calkoxycarbonylxe2x80x9d refers to xe2x80x94C(O)OR wherein R is alkyl.
The term xe2x80x9caralkenylxe2x80x9d refers to an alkenyl group substituted with an aryl group. Preferably the alkenyl group has from 2 to about 6 carbon atoms.
The term xe2x80x9caralkylxe2x80x9d refers to an alkyl group substituted with an aryl group. Suitable aralkyl groups include benzyl, phenethyl, and the like, all of which may be optionally substituted. Preferably the alkyl group has from 1 to about 6 carbon atoms.
The term xe2x80x9carylxe2x80x9d refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes a carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
The term xe2x80x9caryloxyxe2x80x9d refers to a group having the formula, Rxe2x80x94Oxe2x80x94, wherein R is an aryl group.
The term xe2x80x9caralkoxyxe2x80x9d refers to a group having the formula, Rxe2x80x94Oxe2x80x94, wherein R is an aralkyl group.
The term xe2x80x9camino acidxe2x80x9d refers to both natural, unnatural amino acids in their D and L stereo isomers if their structures allow such stereoisomeric forms, and their analogs. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminoisobutyric acid, demosine, 2,2xe2x80x2-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, ornithine and pipecolic acid. Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.
The term xe2x80x9camino acid analogxe2x80x9d refers to an amino acid wherein either the C-terminal carboxy group, the N-terminal amino group or side-chain functional group has been chemically modified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine.
The term xe2x80x9camino acid residuexe2x80x9d refers to radicals having the structure: (1) xe2x80x94C(O)xe2x80x94Rxe2x80x94NHxe2x80x94, wherein R typically is xe2x80x94CH(Rxe2x80x2)xe2x80x94, wherein Rxe2x80x2 is H or a carbon containing substituent; or (2) 
wherein p is 1, 2 or 3 representing the azetidinecarboxylic acid, proline or pipecolic acid residues, respectively.
xe2x80x9cBiarylxe2x80x9d refers to phenyl substituted by carbocyclic or heterocyclic aryl as defined herein, ortho, meta or para to the point of attachment of the phenyl ring.
xe2x80x9cBrinexe2x80x9d refers to an aqueous saturated solution of sodium chloride.
xe2x80x9cCarbocyclic arylxe2x80x9d refers to aromatic groups wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and naphthyl groups, all of which may be optionally substituted. Suitable carbocyclic aryl groups include phenyl and naphthyl. Suitable substituted carbocyclic aryl groups include indene and phenyl substituted by one to two substituents such being advantageously lower alkyl, hydroxy, lower alkoxy, lower alkoxycarbonyl, halogen, trifluoromethyl, difluoromethyl, nitro, and cyano. Substituted naphthyl refers to naphthyl, more preferably 1- or 2-naphthyl, substituted by Y1, Y2 and/or Y3 as defined in connection with formula (I) hereinabove.
xe2x80x9cCycloalkenylxe2x80x9d refers to a cyclic alkenyl group. Suitable cycloalkenyl groups include, for example, cyclopentenyl and cyclohexenyl.
xe2x80x9cCycloalkylxe2x80x9d refers to a cyclic alkyl group having at least one ring and includes polycyclic groups, including fused ring cyclic alkyl groups. Suitable cycloalkyl groups include, for example, cyclohexyl, cyclopropyl, cyclopentyl, and cycloheptyl.
xe2x80x9cCyclohexylmethylxe2x80x9d refers to a cyclohexyl group attached to CH2.
xe2x80x9cFused carbocyclicxe2x80x9d refers to a multicyclic fused carbocyclic ring having both aromatic and non-aromatic rings. Suitable fused carbocyclic rings include fluorenyl, tetralin and the like.
xe2x80x9cFused carbocyclic alkylxe2x80x9d refers to an alkyl group substituted with a fused carbocyclic ring moiety, preferably a multicyclic fused carbocyclic ring including both aromatic and non-aromatic rings. Suitable fused carbocyclic alkyl groups include fluorenylmethyl, and the like.
The term xe2x80x9chalogenxe2x80x9d refers to fluorine, chlorine, bromine and iodine.
xe2x80x9cHeteroaralkenylxe2x80x9d refers to an alkenyl group substituted with a heteroaryl, and includes those heterocyclic systems described in xe2x80x9cHandbook of Chemistry and Physicsxe2x80x9d, 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Preferably the alkenyl group has from 2 to about 6 carbon atoms.
xe2x80x9cHeteroaralkylxe2x80x9d refers to an alkyl group substituted with a heteroaryl, such as picolyl, and includes those heterocyclic systems described in xe2x80x9cHandbook of Chemistry and Physicsxe2x80x9d, 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Preferably the alkyl group has from 1 to about 6 carbon atoms.
xe2x80x9cHeteroarylxe2x80x9d refers to aromatic groups having from 1 to 14 carbon atoms and the remainder of the ring atoms are heteroatoms, and includes those heterocyclic systems described in xe2x80x9cHandbook of Chemistry and Physicsxe2x80x9d, 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Suitable heteroatoms include oxygen, nitrogen, and S(O)i, wherein i is 0, 1 or 2, and suitable heterocyclic aryls include furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, and the like.
xe2x80x9cHeterocycloxe2x80x9d refers to a reduced heterocyclic ring system comprised of carbon, nitrogen, oxygen and/or sulfur atoms, and includes those heterocyclic systems described in xe2x80x9cHandbook of Chemistry and Physicsxe2x80x9d, 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems.
xe2x80x9cHeterocycloalkylxe2x80x9d refers to an alkyl group substituted with a heterocyclo group, and includes those heterocyclic systems described in xe2x80x9cHandbook of Chemistry and Physicsxe2x80x9d, 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Preferably the alkyl group has from about 1 to about 6 carbon atoms.
The term xe2x80x9clowerxe2x80x9d referred to herein in connection with organic radicals or groups defines such radicals or groups with one and up to and including 5 carbon atoms, preferably up to and including 4 carbon atoms, and advantageously one or two carbon atoms. Such radicals or groups may be straight chain or branched chain.
xe2x80x9cPerfluoroalkylxe2x80x9d refers to an alkyl group which has every hydrogen replaced with fluorine.
xe2x80x9cPerfluoroarylxe2x80x9d refers to an aryl group which has every hydrogen replaced with fluorine.
xe2x80x9cPerfluoroarylalkylxe2x80x9d refers to an aralkyl group in which every hydrogen on the aryl moiety is replaced with fluorine.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d includes salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid. In practice the use of the salt form amounts to use of the base form. The compounds of the present invention are useful in both free base and salt form, with both forms being considered as being within the scope of the present invention.
xe2x80x9cAcNxe2x80x9d or xe2x80x9cMeCNxe2x80x9d refers to acetonitrile.
xe2x80x9cAIBNxe2x80x9d refers to 2,2xe2x80x2-azobisisobutyronitrile.
xe2x80x9cBnxe2x80x9d refers to benzyl.
xe2x80x9cBocxe2x80x9d refers to t-butoxycarbonyl.
xe2x80x9cBoc2Oxe2x80x9d refers to Boc anhydride (di-tert-butyl carbonate).
xe2x80x9cBzlSO2xe2x80x9d refers to benzylsulfonyl.
xe2x80x9cCbzxe2x80x9d or xe2x80x9cCBzxe2x80x9d refers to benzyloxycarbonyl.
xe2x80x9cCsCo3xe2x80x9d refers to cesium carbonate.
xe2x80x9cDCAxe2x80x9d refers to dichloroacetic acid.
xe2x80x9cDCCxe2x80x9d refers to N,Nxe2x80x2-dicyclohexylcarbodiimide.
xe2x80x9cDCMxe2x80x9d refers to dichloromethane.
xe2x80x9cDIEAxe2x80x9d refers to diisopropylethylamine.
xe2x80x9cDMFxe2x80x9d refers to N,N-dimethylformamide.
xe2x80x9cDMSOxe2x80x9d refers to dimethyl sulfoxide.
xe2x80x9cDMAPxe2x80x9d refers to 4-N,N-dimethylaminopyridine.
xe2x80x9cEDCxe2x80x9d refers to 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride salt.
xe2x80x9cEt3Nxe2x80x9d or xe2x80x9cTEAxe2x80x9d refers to triethylamine.
xe2x80x9cEtOAcxe2x80x9d refers to ethyl acetate.
xe2x80x9cEtOHxe2x80x9d refers to ethanol.
xe2x80x9cHATUxe2x80x9d refers to O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluromium hexafluorophosphate.
xe2x80x9cHBTUxe2x80x9d refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
xe2x80x9cHClxe2x80x9d refers to hydrochloric acid.
xe2x80x9cHOAcxe2x80x9d refers to acetic acid.
xe2x80x9cHOAtxe2x80x9d or xe2x80x9cHOATxe2x80x9d refers to 1-hydroxy-7-azabenzotriazole.
xe2x80x9cHOBtxe2x80x9d refers to 1-hydroxybenzotriazole monohydrate.
xe2x80x9c1-BuOCOClxe2x80x9d refers to isobutylchloroformate.
xe2x80x9cHPLCxe2x80x9d refers to high pressure liquid chromatography.
xe2x80x9cLiAlH4xe2x80x9d refers to lithium aluminum hydride.
xe2x80x9cLiAlH2(OEt)2xe2x80x9d refers to lithium aluminum hydride diethoxide.
xe2x80x9cMexe2x80x9d refers to methyl.
xe2x80x9cMeOHxe2x80x9d refers to methanol.
xe2x80x9cNMMxe2x80x9d refers to N-methylmorpholine.
xe2x80x9cNBSxe2x80x9d refers to N-bromosuccinimide.
xe2x80x9cPhB(OH)2xe2x80x9d refers to phenylboronic acid.
xe2x80x9cPh3Pxe2x80x9d or xe2x80x9cPPh3xe2x80x9d refers to triphenylphospine.
xe2x80x9cPyBOPxe2x80x9d refers to benzotriazole-ly-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate.
xe2x80x9cRP-HPLCxe2x80x9d refers to reverse phase high pressure liquid chromatography.
xe2x80x9cTFAxe2x80x9d refers to trifluoroacetic acid.
xe2x80x9cTHFxe2x80x9d refers to tetrahydrofuran.
xe2x80x9cTLCxe2x80x9d refers to thin layer chromatography.